chemotherapyinduced immunogenic modulation of tumor …to-5; erp57-speciﬁc mab to-2; and...

Chemotherapy-induced immunogenic modulation of tumorcells enhances killing by cytotoxic T lymphocytes and isdistinct from immunogenic cell death

James W. Hodge1, Charlie T. Garnett1, Benedetto Farsaci1, Claudia Palena1, Kwong-Yok Tsang1,

Soldano Ferrone2 and Sofia R. Gameiro1

1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD2 Cancer Immunology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune

responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast,

several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases

tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model,

we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did

not induce ATP or high-mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all

cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC-1, or PSA-specific CD81 CTLs was

significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing ma-

chinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT-blocking

peptide. A docetaxel-resistant cell line was selected (MDR-11, CD1331) by continuous exposure to docetaxel. These cells, while resist-

ant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL kill-

ing. Here, we provide an operational definition of “immunogenic modulation,” where exposure of tumor cells to nonlethal/sublethal

doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and

complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.

Anticancer therapies such as chemotherapy and radiationaim for direct killing of tumor cells. However, recent studieshave demonstrated that these modalities may have immuno-modulatory effects, through direct action on tumor cells oron cells of the immune system.1,2 Docetaxel, a member of thetaxane family of drugs, exhibits broad antitumor activity bymicrotubule stabilization and is currently indicated for thetreatment of multiple cancer types. We previously reportedthat docetaxel modulated CD41, CD81, CD191, naturalkiller cell and T-regulatory (Treg) populations in nontumor-bearing mice, and enhanced interferon-g (IFN-g) productionby CD81 T cells.2 We also showed that docetaxel combinedwith vaccine increased antigen-specific T-cell responses to theantigen expressed by the vaccine and was superior to eitheragent alone at reducing tumor burden. Here, we examine theability of this taxane to alter the phenotype of human tumorcells and increase their susceptibility to CD81 cytotoxic Tlymphocyte (CTL)-mediated killing. We also provide anoperational definition of “immunogenic modulation,”whereby exposure of tumor cells to nonlethal/sublethal dosesof chemotherapy (due to resistance or low-dose delivery)alters tumor phenotype to render the tumor more sensitiveto CTL killing.

We hypothesized that immunogenic modulation of tumor-cell phenotype and antigen-processing machinery (APM) bydocetaxel could enhance productive interactions between

Key words: docetaxel, immunogenic cell death, immunogenic mod-

ulation, calreticulin, antigen-processing machinery, T-cell response

*Published 2013. This article is a U.S. Government work, and as

such, is in the public domain in the United States of America.

Abbreviations: ABC: ATP-binding cassette; APM: antigen-processing

machinery; CC: criterion of continuity; CEA: carcinoembryonic

antigen; CMA: concanamycin A; CRT: calreticulin; DC: dendritic

cell; ER: endoplasmic reticulum; HMGB: high-mobility group box;

ICD: immunogenic cell death; IFN-g: interferon-g; MFI: mean

fluorescence intensity; MUC: mucin; PBS: phosphate-buffered sa-

line; PSA: prostate-specific antigen; TAA: tumor-associated anti-

gen; UPR: unfolded protein response

Grant sponsors: Intramural Research Program of the Center for

Cancer Research, National Cancer Institute, National Institutes of Health

DOI: 10.1002/ijc.28070

History: Received 6 Sep 2012; Accepted 7 Jan 2013; Online 30 Jan

2013

Correspondence to: James W. Hodge, Laboratory of Tumor

Immunology and Biology, Center for Cancer Research, National Cancer

Institute, National Institutes of Health, 10 Center Drive, Room 8B13,

Bethesda, MD, USA, Tel.: 1301–496-0631,

Fax: 1301–496-2756, E-mail: [email protected]

Dr. Garnett’s current address is: Department of Biology, Biology and

Immunology Program, Georgia State University, Atlanta, Georgia

Dr. Ferrone’s current address is: Department of Surgery, Massachu-

setts General Hospital, Boston, Massachusetts

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Int. J. Cancer: 133, 624–637 (2013) 2013 UICC

International Journal of Cancer

IJC

CD81 CTLs and cancer cells. In this study, we examined theeffects of docetaxel on molecules that have been implicated inenhancing T cell-mediated tumor-cell killing through diversemechanisms, including calreticulin (CRT), Fas, the adhesion/costimulatory molecule ICAM-1, MHC class I, the human tu-mor antigens carcinoembryonic antigen (CEA) and mucin-1(MUC-1), and components of the antigen-processing chain.3

In addition, we determined that cells resistant to the directcytostatic effects of docetaxel were still modified by the drugin a manner that resulted in enhanced lysis by CTLs, com-pared with untreated cells.

These studies are the first to report (i) an evaluation of hall-marks of immunogenic cell death (ICD) and immunogenic mod-ulation following docetaxel treatment of prostate, breast andcolon cancer cell lines, (ii) the use of docetaxel to functionallyenhance antigen-specific CTL-mediated killing, (iii) the use ofdocetaxel to functionally enhance antigen-specific CTL-mediatedkilling of a docetaxel-resistant tumor cell line by modulation ofAPM and (iv) the functional role of CRT in immunogenic mod-ulation. These observations are distinct and complementary tothose of ICD and highlight a mechanism whereby chemotherapycan be used in combination with active immunotherapy.

Material and MethodsTumor cell lines

Cells of human prostate carcinoma (LNCaP), breast carci-noma (MCF-7, MDA-MB-231) and colorectal carcinoma(SW620, LS174T) were obtained from American Type Cul-ture Collection (Manassas, VA) and cultured in media desig-nated by the provider for propagation and maintenance.Cells were incubated at 37�C with 5% CO2.

Chemotherapy

Preparations of docetaxel (TaxotereVR , NDC 0075–8003-01;Sanofi Aventis, Bridgewater, NJ) were diluted to 1 mg/mL inphosphate-buffered saline (PBS). The drug was then furtherdiluted in sterile PBS to a working stock of 10 mg/mL. Forcertain studies, mitoxantrone (1 mM, NDC 61703-343-65;Hospira, Lake Forest, IL) was used as a positive control forICD. Sterile PBS was added to control samples.

Analysis of immunogenic cell death

Cells treated with chemotherapy were harvested and assessed forviability by 7AAD staining and cell-surface expression of CRT(see below). Supernatant fluids were analyzed for high-mobility

group box 1 (HMGB1) protein by ELISA (IBL International,Hamburg, Germany), and for ATP by bioluminescence (Sigma,St. Louis, MO), according to the manufacturer. In all cases, start-ing (Day 0) cell number and media volume were controlledacross all four cell lines and all four assays.

Flow cytometry analysis

Cell-surface staining of tumor cells was performed using theprimary labeled monoclonal antibodies (mAb) CD95-FITC,CD54-PE, CD66-FITC, CD227-FITC, HLA-ABC-PE (BDBiosciences, San Diego, CA) and CRT-PE (R&D Systems,Minneapolis, MN). The appropriate isotype-matched controlswere purchased from BD Biosciences. CD133-APC was pur-chased from Miltenyi Biotech (Auburn, CA). MDR-1(ABCB1) was purchased from eBioscience (San Diego, CA).Proteins were scored as upregulated if detection levelsincreased by �10% or if mean fluorescence intensity (MFI)increased by� 30% following docetaxel treatment. Stainedcells were acquired on a FACScan or FacsCalibur flow cy-tometer using CellQuest software (BD Biosciences). Isotypecontrol staining was <5% for all samples analyzed. For analy-sis of APM, LMP2-specific mAb SY-1; TAP-1-specific mAbNOB1; TAP-2-specific mAb NOB2; calnexin-specific mAbTO-5; ERp57-specific mAb TO-2; and tapasin-specific mAbTO-3 were developed and characterized as described.4 MouseIgG1 mAb isotype control was obtained from BD Bioscien-ces. Cell surface and intracytoplasmic staining of cells wasperformed as described.5

In vivo studies

Studies were conducted with 6- to 8-week-old female nudemice (nu/nu) (Charles River, Wilmington, MA). LNCaP tu-mor cells (8 3 106) were admixed 1:1 with Matrigel (BD Bio-sciences) and injected s.c. When tumors reached a volume of1,000 mm3, mice were given 0.5 mg of docetaxel or PBS i.p.once every other day over a period of 5 days, equivalent to75–100 mg/m2. On Day 7, tumors were surgically removedand examined by flow cytometry and histochemistry. For flowcytometry, tumors were processed into single-cell suspensionsand staining was performed as described above. For immuno-histochemistry, fresh tissues were immersed in the bufferedzinc fixative Z-fix (Anatech LTD, Battle Creek, MI) and em-bedded. 5-lm paraffin sections were cut and incubated withserum block, followed by anti-CEA mAb (COL-1),6 anti-MUC-1 mAb (DF-3),7 anti-CRT (clone FMC75, Abcam,Cambridge, MA) or isotype-matched control antibodies,

What’s new?

Some chemotherapies not only kill cancer cells, but also enhance immune responses against those cells. In this study, the

authors found that when tumor cells were exposed to nonlethal doses of docetaxel, they became more sensitive to killing by

cytotoxic T lymphocytes. This process appears to be mediated by a number of molecules, including calreticulin. Even tumor

cells that were resistant to docetaxel became more susceptible to lysis by CTLs. These results suggest that chemotherapy

combined with immunotherapy might improve outcomes in patients who have failed chemotherapy alone.

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Hodge et al. 625

Int. J. Cancer: 133, 624–637 (2013) VC 2013 UICC

followed by ABC peroxidase (Vector Labs, Burlingame, CA)and diaminobenzidine. The sections were rinsed and counter-stained with hematoxylin. The entire slides were digitallyscanned by an Aperio ScanScope CS scanning system (AperioTechnologies, Vista, CA) and analyzed by the Aperio Image-Scope Viewer software. Necrotic regions were excluded fromanalysis. The positive pixel count v9 algorithm was used tomeasure CEA1, MUC-11 or CRT1 tumor regions. For CRTmembrane staining, the membrane algorithm was used.

CTL lines

The HLA-A2-restricted, CEA-specific, CD81 cytotoxic T-cellline (designated CEA CTL) recognizes the CEA peptide epi-tope YLSGANLNL (CAP-1).8 It was maintained and propa-gated as previously described.9 The HLA-A2-restricted,prostate-specific antigen (PSA)-specific, CD81 CTL line (des-ignated PSA CTL) recognizes the PSA peptide epitopeVLSNDVCAQV.7 The HLA-A2-restricted, MUC-1-specific,CD81 CTL line (designated MUC-1 CTL) recognizes theMUC-1 peptide epitope ALWGQDVTSV.7

Cytotoxicity assays

CTLs specific for CEA, PSA, or MUC-1 were used on Day 4of the restimulation cycle following Ficoll purification. Doce-taxel-treated and untreated human tumor cells were culturedwith drug for 72 hr and subsequently used as targets in astandard cytotoxicity assay using 111In.1,10 Radiolabeled tumorcells (2 3 103) were incubated with 6 3 104 antigen-specificCTLs (E:T ratio of 30:1) for 18 hr at 37�C with 5% CO2. Tar-gets and CTLs were suspended in complete medium supple-mented with 10% human AB serum in 96-well U-bottomplates (Costar, Cambridge, MA). After incubation, superna-tants were collected and assayed on a Cobra Autogamma(Packard Instruments, Downers Grove, IL). The percentage ofspecific lysis was determined by the standard equation: % spe-cific lysis5 [(experimental-spontaneous)/(maximum-spontane-ous)] 3 100. For MHC-I-blocking studies, tumor cells wereincubated with anti-HLA-A2 mAb (20 mg/mL, AbD Serotec,Raleigh, NC) or isotype control mAb (IgG2b, 20 mg/mL, AbDSerotec) for 1 hr at 37�C before being used as CTLs. Targettumor cells were then incubated with CTLs, as above. Forindicated experiments, CEA-specific CTLs were preincubatedfor 2 hr in the presence of 100 nM concanamycin A (CMA)to specifically inhibit perforin-dependent lysis, and incubatedwith target cells; CMA was present during the assay. For indi-cated experiments, the CTL assay was performed in the pres-ence of CEA peptide or a CRT-blocking peptide (0.17 mM,MBL International, Woburn, MA). As a control, a 15 merpeptide, LCMV118–132 (RPQASGVYMGNLTAQ) was used.

RNA isolation, real-time PCR and siRNA knockdown(s)

For selected cell lines, cells were seeded in T-75 flasks at 0.5–13 107 cells/flask. Cells were allowed to adhere for 12 hr, thentreated with either 25 or 250 ng/mL docetaxel or left untreated.Real-time PCR analysis was performed on cDNA isolated from

docetaxel-treated or untreated cells using TaqMan Gene Expres-sion Master Mix (Applied Biosystems, Carlsbad, CA). The pri-mers used to amplify the genes of interest were Hs00189032_m1(h-CRT), Hs00984006_m1 (h-PERK) and human GAPDH en-dogenous control (Applied Biosystems). Gene expressions wereobtained after 40 light cycles using a 7300 Real-Time PCRmachine (Applied Biosystems) using the following cycle condi-tions: 2 min at 50�C and 10 min at 95�C, followed by 40 cyclesof 15 sec at 95�C and 1 min at 60�C. Experiments were per-formed in triplicate. Data were analyzed and genes were normal-ized against the housekeeping gene GAPDH. For tumor-cellsilencing of CRT or PERK, siRNA and negative control siRNAwere used according to the manufacturer’s instructions (SilencersiRNA, Life Technologies, Grand Island, NY).

Generation of a docetaxel-resistant tumor cell line

SW620 cells were made resistant to docetaxel as previouslydescribed.11 Briefly, cells were passaged in increasing concen-trations of docetaxel to generate docetaxel resistance. Thedrug concentration was increased three-fold every 2 weeks(two passages). Cells able to tolerate the highest concentra-tion of drug (3,000 ng/mL) and continue to proliferate wereused for subsequent studies.

Statistical analysis

Tests of significance are reported as p values, derived fromStudent’s t-test using a two-tailed distribution and calculatedat 95% confidence using GraphPad Prism 4.0 for Macintosh.Significant differences in the distribution of flow cytometryanalysis data were determined by the Kolmogorov-Smirnovtest, using CellQuest software (BD Biosciences).

ResultsTumors treated with docetaxel increase CRT surface

expression but fail to undergo immunogenic cell death

Certain chemotherapeutic agents can stimulate immunogenictumor cell death.12 The cardinal signs of ICD are (i) CRT ex-posure on the surface of dying cells, (ii) the release ofHMGB1, (iii) the release of ATP, and most importantly, (iv)cell death.13 Each of these molecules acts on dendritic cells(DCs) to facilitate the presentation of tumor antigens to theimmune system.14 CRT, a chaperone and calcium regulator,when exposed on the surface of a dying cell, serves as aphagocytic signal to DCs.15,16 When HMGB1, a nonhistonechromatin-binding protein, is released from dying cells, itengages TLR-4 on DCs, leading to maturation.13 The secre-tion of ATP by dying cells binds purigenic PRX7 receptorson DCs, further supporting T-cell activation.12

We first examined whether in vitro treatment with therapeu-tic doses of docetaxel induced ICD in a panel of four human car-cinoma cell lines (one prostate, two breast, one colorectal). Cellswere subjected to 0–3,500 ng/mL of docetaxel for 72 hr. Mitox-antrone was used to induce ICD as a positive control.12 Treat-ment of LNCaP tumor cells with docetaxel significantly inducedtranslocation of CRT to the cell surface in a dose-dependent

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626 Chemotherapy induces immunogenic modulation


manner (Fig. 1a). However, docetaxel treatment did not result inthe secretion of HMGB1 (Fig. 1b) or ATP at any concentration(Fig. 1c). Finally, treatment of these tumor cells with docetaxeldid not induce cell death at 2.5–250 ng/mL; however, at veryhigh concentrations of docetaxel (3,500 ng/mL), cells displayedsignificantly decreased viability as determined by 7AAD staining.Similar results were observed with the breast cancer lines MCF-7and MDA-MB-231, and with the colon cancer cell line SW620(Figs. 1a–1d). For each cell line, treatment with mitoxantroneunequivocally induced all four molecular determinants of ICD.Taken together, these results show that docetaxel treatment,while significantly modulating CRT translocation, fails to induceclassic ICD.

Tumor cells treated with chemotherapy undergo

immunogenic modulation and demonstrate significantly

increased sensitivity to antigen-specific cytotoxic T-cell

killing

As several cell-surface proteins on tumor target cells havepreviously been demonstrated to be critical for interactionswith CD81 T cells,1 we examined the potential role of altered

tumor phenotype on CTL sensitivity (immunogenic modula-tion). Cells subjected to docetaxel were analyzed for surfaceexpression of Fas, ICAM-1, CEA, MUC-1 and MHC-I. CRTwas also monitored by flow cytometry. While this chemo-therapy treatment was nonlytic, there were notable alterationsin expression of the surface proteins analyzed. Markedincreased expression of CEA and CRT was the most com-monly observed change, with all (4/4) cell lines increasingsurface expression of each molecule (Fig. 2a). Upregulationof MUC-1 and Fas (2/4 cell lines) was also observed. In addi-tion, treatment of LNCaP tumor cells with docetaxel signifi-cantly induced upregulation of other prostate tumor antigensas determined by RT-PCR: PSA, 1.34-fold increase, prostatestem cell antigen (PSCA), 1.89-fold increase, prostate-specificmembrane antigen (PSMA), 1.28-fold increase and PAP,1.46-fold increase (data not shown).

To determine the functional significance of cellular altera-tions induced by docetaxel, tumor cell lines were treated andcoincubated with the CEA-, PSA- and/or MUC-1-specificCTL. Untreated LNCaP cells were killed with CEA-specificT cells at a level of 8%. Docetaxel treatment of these

Figure 1. Tumor cells treated with docetaxel show increased surface expression of CRT, but do not undergo ICD. Four human tumor cell

lines were treated with 2.5–250 ng/mL (black bars), or 3,500 ng/mL docetaxel (open bars). Mitoxantrone (1 mM) was used as a positive

control (crosshatched bars). After 72 hr of incubation, cells were examined for cardinal signs of ICD. (a) Surface expression of CRT. (b)

HMGB1 secretion. (c) ATP secretion. (d) Percentage of dying cells (7AAD1). * 5 statistical significance relative to untreated cells. This

experiment was repeated two times with similar results.

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Figure 2. Tumor cells treated with a chemotherapeutic agent undergo immunogenic modulation and demonstrate significantly increased

sensitivity to antigen-specific CTL killing. (a) Human tumor cells were treated in vitro for 72 hr with 2.5, 25 or 250 ng/mL of docetaxel, or

left untreated. Cells were analyzed after each treatment for surface expression Fas, ICAM-1, CEA, MUC-1, MHC-I and CRT. Numbers indicate

percentage of positive cells. Numbers in parentheses denote MFI. Bold type indicates marked upregulation (�10% increase in percent of

cells or 30% increase in MFI not observed in isotype control vs. untreated cells). (b) Human tumor cells treated in vitro for 72 hr with 25

(white bars) or 250 (black bars) ng/mL of docetaxel, or left untreated (gray bars), were used as targets in an 18-hr CTL lysis assay. CEA-,

PSA- or MUC-1-specific CD81 T cells were used as effector cells at an E:T ratio of 30:1. For controls, tumor cells were incubated with anti-

HLA-A2 mAb or CMA. CEA1HLA-A2– LS174T cells were used to verify CTL specificity. ND: not determined. * 5 statistical significance relative

to untreated cells. This experiment was repeated four times with similar results.

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cells substantially increased CTL killing in a dose-dependentmanner (19% and 42%, Fig. 2b, p5< 0.0001 vs. no treat-ment for both 25 and 250 ng/mL). Moreover, MCF-7, MDA-MB-231 and SW620 tumor cells also demonstrated signifi-cantly increased sensitivity to CEA-specific killing after doce-taxel treatment (Fig. 2b). In addition, LNCaP tumor cellswere killed at significantly greater levels by PSA-specificCTLs, and MCF-7 tumor cells were killed by MUC-1-specificT cells following docetaxel treatment. These data indicatethat exposure of cells to docetaxel enhances antigen-specificCTL-mediated killing of tumor cells, and that this effect canbe extended to a variety of tumor-associated antigens(TAAs). The CTL killing was MHC-restricted as determinedby anti-MHC (HLA-A2) blocking antibody (Fig. 2b), andperforin mediated, as CMA-treated CTLs (which abrogatesperforin-based cytotoxic activity) showed significantly

reduced killing (p 5 0.01) (Fig. 2b). In addition, CEA1,HLA-A2–, LS174T cells were used to verify CTL specificity.This experiment was repeated four times with similar results.Immunogenic modulation was also observed in cells treatedwith another taxane, paclitaxel. For LNCaP prostate tumorcells treated with 3.65 mg/mL paclitaxel (equivalent to 175mg/m2),17 CEA-specific killing increased 3.2-fold (116 6%killing in untreated or cremophor-treated cells vs. 366 12%in paclitaxel-treated cells, p 5 0.032, data not shown).

Tumors treated in vivo with docetaxel modulate

tumor phenotype

To confirm phenotypic changes induced by docetaxel in vivo,nude mice were implanted with LNCaP tumors and treatedwith docetaxel or PBS. Prostate tumor cells increased expres-sion of the TAAs CEA and MUC-1 1.5-fold after docetaxel

Figure 3. In vivo treatment with docetaxel modulates tumor phenotype. Nude mice bearing LNCaP xenografts were treated with docetaxel or

vehicle (PBS). One week later, tumors were surgically removed, stained and evaluated by immunohistochemistry for expression of the tumor

antigens CEA or MUC-1 (a, 403, inset: isotype control), or CRT (b, 203, inset 403). Numbers indicate percentage of positive cells as deter-

mined by pixel analysis (n 5 2 mice/treatment group). Arrows indicate CRT membrane staining. (c) Flow cytometry of tumors treated with

docetaxel (open histograms) or PBS (shaded histograms). Numbers indicate percentage of positive cells. Numbers in parentheses denote

MFI. * 5 statistical significance relative to untreated cells. Tum

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Hodge et al. 629


treatment (p5 0.01) (Fig. 3a). CRT is heterogeneouslyexpressed in untreated LNCaP cells. As seen in Figure 3b,12% of CRT cells stained strongly positive, and cellular local-ization of CRT was diffuse (inset panel). After treatment withdocetaxel, however, there was a sharp and significant increasein expression of CRT, with 26% of cells staining stronglypositive (p5 0.03). Moreover, CRT expression in tumorstreated with docetaxel was associated with the cell membrane(inset, black arrow). In untreated tumors, CRT membranestaining was 2% at 31, 9.6% at 21 and 2.2% at 11 versusdocetaxel-treated tumors, where CRT membrane staining was3.7% at 31, 7.6% at 21 and 4.3% at 11. We next sought toquantify the expression of Fas, ICAM-1, CEA, MUC-1 andMHC-I by flow cytometry (Fig. 3c). LNCaP tumorsresponded to docetaxel with a significant increase (1.4-fold)in expression of Fas, CEA and MUC-1. The increase in CEAand MUC-1 expression corroborated the increase observedby immunohistochemistry (Fig. 3a) and in vitro analysis oftumor cells (Fig. 2a).

Docetaxel-resistant tumor cells undergo immunogenic

modulation and are killed by antigen-specific CTLs at

significantly greater levels after treatment with docetaxel

Human carcinomas often become resistant to the cytotoxiceffects of chemotherapy.18,19 We selected the most docetaxel-resistant SW620 cells present in cultures by serially passingthe cells through increasing concentrations of docetaxel (upto 3,000 ng/mL) to generate a docetaxel-resistant subline.Subsequently, SW620 cells and docetaxel-resistant SW620cells were treated with docetaxel for up to 96 hr. While bothcell lines remained viable in the presence of 250 ng/mL ofdocetaxel, the docetaxel-resistant subline proliferated at levelssimilar to untreated cells (Fig. 4a). The docetaxel-resistantphenotype of these cells was confirmed by the increasedexpression of MDR-1, a member of the ATP-binding cassette(ABC) family of transporters (Fig. 4b). SW620 cells have alsobeen reported to contain a resident subpopulation thatexpresses CD133, which is associated with cancer stem cellsthat can be selected by chemotherapy.20 Here, we found that38% of SW620 cells were CD1331 (Fig. 4b), and that thispercentage increased to 60% in docetaxel-resistant SW620cells.

Although the docetaxel-resistant SW620 cells were resist-ant to the direct inhibitory effects of docetaxel (Fig. 4a), thephenotype of these cells was modulated after docetaxel treat-ment to levels similar to those of the parental SW620 tumorcells (Fig. 4c). Both SW620 and docetaxel-resistant SW620cells showed increased levels of Fas (>9-fold), ICAM-1 (>2-fold) and CEA (>3-fold). These cells also upregulated MUC-1 and MHC-I to similar levels. Furthermore, docetaxel-resist-ant cells, when exposed to docetaxel, exhibited a two-foldincrease in sensitivity to CEA-specific CTL lysis (Fig. 4d,p5 0.001). Lysis by MUC-1-specific CTLs was also signifi-cantly enhanced in docetaxel-resistant SW620 cells treatedwith docetaxel (Fig. 4d, p5 0.0002). The increased CTL sen-

sitivity was similar to the killing seen with untreated anddocetaxel-treated parental SW620 cells (Fig. 4d). These datasuggest that cells insensitive to the cytostatic effects of doce-taxel can nonetheless be sensitized to CTL lysis by exposureto the drug. To confirm that these cells had functionalexpression of MHC-I, SW620 cells and docetaxel-resistantSW620 cells were treated with docetaxel and used in a CEA-specific CTL assay with exogenously loaded CEA peptide(Fig. 4e). As before (Fig. 4d), SW620 cells were killed at a sig-nificantly greater level after treatment with docetaxel. How-ever, untreated cells were killed at a level similar todocetaxel-treated cells when loaded with CEA peptide. Simi-larly, killing of docetaxel-resistant SW620 cells was signifi-cantly enhanced after docetaxel treatment, and peptide-pulsed tumor cells were killed at this similar level.

These data indicate that these cells may have baselineAPM differences that alter CTL antigen presentation of thepeptide required for CTL recognition and killing.

Tumor cells modulate components of the APM

chain after treatment with docetaxel

Components of the APM chain cooperate to present appro-priate peptide epitopes in the context of MHC-I on the sur-face of tumor cells. Defects in APM components on tumorcells have a negative impact on T-cell recognition.21–23 Toexamine the effect of chemotherapy treatment on antigen/MHC-I loading, LNCaP, MCF-7, MDA-MB-231, SW620 anddocetaxel-resistant SW620 cells were treated with docetaxel.Cells were then harvested and examined for modulation andtranslocation of six APM components (Fig. 5a). In all 5 tu-mor cell lines, treatment with docetaxel increased the expres-sion of one or more components of the APM, mostcommonly TAP2 (3/4 tested lines) and calnexin (3/5 lines)(Fig. 5b). These data indicate that chemotherapy-induced up-regulation of APM components, which could result inenhanced loading of MHC-I molecules.

Docetaxel-mediated immunogenic modulation is reduced

in the absence of CRT or PERK, and is reduced by CRT

blocking peptide

CRT, in addition to being modulated by docetaxel treatment(Figs. 1a, 2b, 3a and 4c), is a critical component of antigenprocessing and loading of MHC-I. We thus focused on threeapproaches to determine the functional role of CRT in im-munogenic modulation and subsequent sensitivity to CTL-mediated killing: (i) knockdown of CRT by siRNA, (ii)knockdown of the serine/threonine kinase PERK, which nor-mally phosphorylates EIF2a, culminating in the exposure ofCRT on the cell surface24,25 and (iii) use of a CRT blockingpeptide.26 Knockdown of CRT would normally block APMfunctions, while silencing of PERK would preserve APMfunctions but prevent CRT translocation. Peptide blockingwould prevent surface CRT/T-cell interactions. LNCaP sur-face expression of CRT or PERK was reduced by 84% and88%, respectively (data not shown), following siRNA

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silencing (Fig. 6a). Treatment of these cells with docetaxelresulted in a 60% decrease in upregulated CRT (Fig. 6b). Asbefore, LNCaP cells were killed by CEA-specific T cells to a

significantly greater level after docetaxel treatment (Fig. 6b).However, the level of increased killing diminished signifi-cantly (>3-fold) in LNCaP cells exhibiting reduced

Figure 4. Docetaxel-resistant tumor cells undergo immunogenic modulation and are killed by antigen-specific CTLs at significantly greater

levels after treatment with docetaxel. SW620 cells and SW620 cells passaged in increasing concentrations of docetaxel (designated doce-

taxel-resistant) were treated with docetaxel for up to 96 hr. (a) Tumor cell proliferation was measured after treatment with 0 (closed circles)

or 250 (open squares) ng/mL of docetaxel. Viable cells as determined by trypan blue exclusion were reported after 1, 2, 3 and 4 days of

continuous exposure to docetaxel. * 5 statistical significance. (b) Expression of multidrug-resistance pump MDR-1 and stem cell marker

CD133 on SW620 and docetaxel-resistant SW620 cells. (c) SW620 and docetaxel-resistant SW620 cells were treated in vitro for 72 hr with

250 ng/mL of docetaxel (open histograms) or left untreated (shaded histograms), then analyzed by flow cytometry. Numbers indicate per-

centage of positive cells. Numbers in parentheses denote MFI. * 5 statistical significance relative to untreated cells. (d) CEA- and MUC-1-

specific CTL lysis. Cells incubated for 72 hr with 0 (gray bars) or 250 (black bars) ng/mL of docetaxel were used as targets in an 18-hr CTL

lysis assay, using either CEA-specific or MUC-1-specific CD81 T cells as effector cells. (e) Target tumor cells were pulsed with CEA peptide

at an E:T ratio of 30:1. * 5 statistical significance. This experiment was repeated three times with similar results.

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Figure 5. Tumor cells modulate components of the APM chain after treatment with docetaxel. (a) Schematic of antigen-processing compo-

nents from cytoplasm to cell surface. Arrows indicate functional checkpoints for CRT and PERK. (b) Human tumor cells were treated in vitro

for 72 hr with 2.5–250 ng/mL of docetaxel or left untreated, then analyzed for key intracellular components of the APM chain by flow

cytometry. Numbers indicate percentage of positive cells. Numbers in parentheses denote MFI. Bold type indicates marked upregulation

(�10% increase in percent of cells or 30% increase in MFI not observed in isotype control vs. untreated cells).

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expression of CRT or PERK. The reduction in CTL killingwas not observed in cells treated with control siRNA. Finally,LNCaP cells incubated with control blocking peptide werekilled to a significantly greater level after docetaxel treatment(Fig. 6c). However, in LNCaP cells coincubated with the CRTblocking peptide, docetaxel-enhanced PSA-specific killing ofLNCaP cells was reduced (Fig. 6c). As a control, incubationof the CEA-specific CTL used in these assays with CRTblocking peptide did not decrease T-cell viability (notshown).

DiscussionZitvogel and Kroemer et al. have elegantly shown that certainchemotherapeutic regimens trigger cancer cell death whilestimulating endogenous immune responses against the tu-mor.12–14,16,27–29 Chemotherapy-induced ICD has thus farbeen restricted to only four drugs: cyclophosphamide, doxor-ubicin, oxaliplatin and mitoxantrone.30 However, manychemotherapeutic agents, including cisplatin, 5-fluorouraciland vinorelbine, can upregulate multiple surface moleculeson tumor cells, rendering them more sensitive to immune-mediated killing,2,10,31 without inducing classic ICD.32–35 Thefact that, in many cases, tumor cells do not die after treat-ment led us to investigate the fate of tumor cells that do notdie directly from chemotherapy exposure or from ICD, usingdocetaxel as a model chemotherapeutic agent. ICD was ini-tially characterized by Casares et al., who showed that immu-nizing mice with chemotherapy-treated tumor cells protectedmice from subsequent challenge.36 For our studies usinghuman tumor cells, the term ‘immunogenic modulation’ isderived from the criterion of continuity (CC) definition ofimmunogenicity, where immune cells respond to abruptmodifications of the antigenic patterns with which they arein contact.37 We first examined whether docetaxel couldinduce ICD in four tumor cell lines and found that onlyCRT translocation was induced after docetaxel treatment(2.5–250 ng/mL, Fig. 1). The doses of docetaxel used herewere within the range expected to be administered to patients(100 mg/m2 dose; Cmax of 0.576–3.53 mg/mL, or 668–4095nM plasma concentration).38 At the highest concentration(Cmax), docetaxel-treated cells displayed significantly reducedviability; however, CRT translocation, HMGB1 and ATPsecretion were not observed. This suggested that an alterna-tive mechanism was active in this type of cell death. Doce-taxel has been shown to induce cell death through a non-apoptotic mitotic catastrophe; cells have aberrant mitosis,chromosomal nuclear envelopes, and form large non-viablecells with multiple micronuclei, yet remain intact.39 We pre-viously demonstrated in CEA transgenic mice that docetaxeltreatment of MC38-CEA tumors, which did not impact tu-mor growth, nevertheless induced immune responses to mul-tiple TAAs and resulted in synergistic antitumor activitywhen combined with vaccine.2 It was subsequently deter-mined that MC38-CEA murine tumor cells modulated sur-face expression of several molecules after in vitro treatmentwith 250 ng/mL docetaxel, including Fas (44% positive beforetreatment, 69% positive after treatment) and MHC-1/H2Db

(32% positive before treatment, 72% positive after treatment).In addition, docetaxel-treated tumor cells were killed at amuch greater level by CEA-specific CTLs (30% lysis inuntreated cells vs. 81% lysis in docetaxel-treated cells), (datanot shown). Taken together, these data support the role ofdocetaxel induced immunogenic modulation in vivo, and theenhanced subsequent antitumor effects of antigen-specificT cells induced from a vaccine. This suggested that docetaxel,while not inducing ICD, may nonetheless increase the immu-nogenicity of treated tumor cells. Reports have indicated that

Figure 6. Docetaxel-mediated immunogenic modulation is reduced

in the absence of CRT or PERK and by CRT blocking peptide. (a)

Knockdown of CRT or PERK in LNCaP tumor cells. (b) LNCaP tumor

cells lacking CRT or PERK were treated with 0 (white bars) or 250

(black bars) ng/mL of docetaxel and used as targets in an 18-hr CTL

lysis assay using CEA-specific CD81 T cells. CRT expression was con-

firmed by flow cytometry. Numbers indicate percentage of positive

cells. Numbers in parentheses denote MFI. Bold type indicates

marked upregulation (�10% increase in percent of cells or 30%

increase in MFI not observed in isotype control vs. untreated cells).

(c) LNCaP tumor cells were treated with 0 (white bars) or 250 (black

bars) ng/mL of docetaxel and used as targets in an 18-hr CTL lysis

assay using either CEA-specific or PSA-specific CD81 T cells, in the

presence or absence of CRT blocking peptide (0.17 mM). All assays

were done at an E:T ratio of 30:1. * 5 statistical significance. This

experiment was repeated two times with similar results.Tum

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Hodge et al. 633


induction of ICD depends not on the dose or time of exposureto a given therapeutic, but on the activation of an endoplasmicreticulum (ER) stress module.27 For example, it has beenreported that cisplatin does not induce ICD regardless of dose.However, if tumor cells are treated with cisplatin and thapsigar-gin, an inducer of ER stress, ICD is restored.28 In response to ERstress, the evolutionarily conserved quality control mechanismunfolded protein response (UPR) is triggered with the aim ofrestoring cellular homeostasis during which several ER chaper-ones can translocate to the cell surface, including CRT.40 Certainanticancer therapies, whereas not inducing ICD, may modulateimmune-relevant pathways in the surviving tumor cells as adirect result of UPR adaptive mechanisms to cellular stress.

We next examined whether docetaxel could alter theexpression of six molecules key to T cell-mediated lysis (Fig.2a). In this study, CRT and the TAAs MUC-1 and CEA,which are important T-cell targets, were most often upregu-lated. These phenotypic changes were confirmed in doce-taxel-treated nude mice bearing prostate tumor xenografts(Fig. 3). The mechanism for TAA upregulation followingdocetaxel treatment remains under investigation, focusing onthe above-described UPR. Overexpression of TAAs in tumortissue could result in increased signal 1, making tumor cellsbetter targets of antigen-specific CTLs. We also report here forthe first time the use of docetaxel to functionally enhance anti-gen-specific CTL-mediated killing in human tumor cell lines.All (4/4) docetaxel-treated tumor cells lines were killed at sig-nificantly greater levels by CEA-, MUC-1- and/or PSA-specificHLA-A2-restricted CD81 CTLs than their untreated counter-parts (Fig. 2). CTL activity was abrogated by CMA, indicatingthat the killing was perforin/granzyme-based (Fig. 2b).

Drug resistance, estimated to cause treatment failure in>90% of patients with metastatic disease, is a major consid-eration in initial treatment and in the adjuvant setting. Inthis study, we investigated whether SW620 cells (a line estab-lished from a patient with stage III colorectal carcinoma41)could be selected for enhanced resistance to docetaxel.Although docetaxel’s ability to inhibit proliferation in theseselected cells was abrogated (Fig. 4a), these docetaxel-resist-ant cells continued to modulate cell-surface phenotype inresponse to docetaxel treatment (Fig. 4c). Moreover, thesedocetaxel-resistant cells maintained their sensitivity to lysisby antigen-specific CTLs following docetaxel treatment (Fig.4d). Thus, this study is the first to demonstrate that humantumor cells resistant to the cytostatic effects of docetaxel canstill be immunogenically modulated to enhance sensitivity toimmune-mediated killing in response to drug exposure.These studies complement those of Shtil et al.,42 who trans-fected treated murine tumors with the MDR-1 gene to conferdrug resistance. In vivo, however, chemotherapy-resistanttumors were killed equally well by T cells induced from vac-cination with a whole tumor cell vaccine, showing that thedrug-resistant phenotype did not interfere with lysis by pore-forming CTLs. Our findings are similar, in that cells resistantto docetaxel (Fig. 4) remained sensitive to CTL killing via the

perforin pathway (Fig. 2). Furthermore, SW620 tumor cells,when pulsed with exogenous CEA peptide, were capable ofbeing killed by a CEA-specific CTL to levels observed with doce-taxel-treated tumor cells (Fig. 5e), indicating that the MHCcomplex was structurally intact. We hypothesized that indolentdefects in antigen processing, loading and/or presentation couldbe influenced by exposure to docetaxel. Components of theAPM chain cooperate to present appropriate peptide epitopes inthe context of MHC-I on the surface of tumor cells. APM com-ponents include (from cytoplasm to surface, Fig. 5a) proteosomesubunits LMP-2, peptide transporters TAP-1, TAP-2, chaper-ones calnexin and CRT, peptide loaders Erp57 and tapasin andHLA.3 APM defects have been described in several tumors ofdifferent histologies, as well as in established cell lines,43,44 andhave been reported to have a negative impact on T-cell recogni-tion.22,23 Exposure of tumor cells to docetaxel resulted in sub-stantial upregulation of one or more APM components, whichwould positively impact the loading of MHC. Modulation ofAPM components by docetaxel was also observed in tumor cellsresistant to docetaxel (SW620-TR, Fig. 5b). These data extendthe findings of two other investigators: Setiadi et al., whoreported that histone deacetylase inhibitors also modulatedAPM,23 including TAP-1, TAP-2, LMP-2 and tapasin; andLopez-Albaitero et al.,22 who described several levels of APMdefects that interfered with CTL recognition and killing in squa-mous cell carcinoma of the head and neck. Treating APM com-ponents in these cells with IFN-g restored CTL sensitivity.

CRT is a major Ca21 binding protein, normally located inthe lumen of the ER, that binds to misfolded proteins andacts as a molecular chaperone. Emerging data suggest thatCRT expressed on the surface of cells undergoing ICD alsoacts as a phagocytic signal.15,16 CRT is closely associated withMHC-I and is an important peptide-loading complex. If littleor no CRT is present, MHC-I molecules are still able toappear on the cell surface, but the majority are loaded withsuboptimal peptides.45 Most importantly, Fu et al. havereported that CRT maintains the low threshold of peptiderequired for efficient antigen presentation. Given that CRTtranslocation was observed in docetaxel-treated cells (Figs. 1,2a, 3b and 4c), and that CRT enriches endogenous peptidesin the ER, which is critical for efficient antigen presenta-tion,45 we next investigated the role of CRT in immunogenicmodulation. CTL killing of docetaxel-treated tumor cells waslargely inhibited following CRT knockdown (Fig. 6b). Thetranslocation of CRT from the cytoplasm to the cell surfacehas been shown to be mainly governed by PERK-mediatedphosphorylation of the translation initiation factor eIF2a.24

Knockdown of PERK in our model significantly interferedwith the increased level of CTL killing observed after doce-taxel treatment (Fig. 6b). These data are consistent with thefindings of Garg et al., who reported that the absence ofPERK following shRNA knockdown in CT26 murine tumorcells compromised the ability of mitoxantrone-treated cells tosecrete ATP and translocate CRT to the cell surface.46

Finally, because CTL killing was abrogated in the presence ofa CRT blocking peptide (Fig. 6c), CTL killing of docetaxel-

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treated tumor cells was determined to be dependent on CRT’sinteraction with CTLs. Low-density lipoprotein receptor-relatedprotein 1 (LRP-1/CD91) has been identified as a potential candi-date receptor for CRT by binding interaction analysis.46 In addi-tion, Li et al. have shown that activation of lymphocytespromotes cell-surface expression of thrombospondin-1, whichbinds to LPR-1/CD91 and CRT.47 However, the CEA-specific Tcells used in these studies did not express LPR-1/CD91 (datanot shown). Additional CRT receptors on T cells and/or DCsneed to be identified16; candidates include scavenger receptor Aand scavenger receptor class F member 2.15

CRT exposure has been shown to be an important factorfor the uptake of tumor cells by DCs or the phagocytosis ofapoptotic cells. However, it is unclear whether docetaxel treat-ment may also affect these processes. To examine this, humanPBMC derived immature DCs (GM-CSF/IL-4) were coincu-bated with LNCaP tumor cells or LNCaP tumor cells treatedfor 48 hr with 250ng/mL docetaxel and subsequently examinedby flow cytometry for DC maturation markers as an indirectindication of tumor cell phagocytosis. As a positive control,DCs were matured with CD40L. DCs incubated with LNCaPcells, or LNCaP cells treated with docetaxel failed to undergomaturation as measured by increased expression of CD80,CD83, CD54, CD58, HLA-ABC and HLA-DR/DP/DQ (datanot shown). These data suggest that docetaxel-treated tumorcells may interact primarily with CD81 T cells for direct tumorcell killing and that the DCs in the environment may play agreater role in the uptake of cellular debris resulting in theinduction of T-cell responses to multiple tumor antigens (anti-gen cascade/epitope spreading).

We propose here that immunogenic modulation of tu-mor cell phenotype (Figs. 1, 2a, 3 and 4c) and APM (Fig.5b) by sublethal chemotherapy enhances productive interac-tions between CTLs and cancer cells, resulting in enhancedtumor cell killing (Figs. 2b, 4d, 6b and 6c). The data pre-

sented here suggest that optimal tumor therapies are thosethat can capitalize on the changes induced by conventionaltherapies to achieve synergy with immunotherapies. Inpatients, it is possible that treatment of tumors with chemo-therapy could induce a continuum of responses rangingfrom necrosis, “classic” ICD, non-classical ICD, to immuno-genic modulation. Indeed, in clinical trials docetaxel hasshown evidence of clinical benefit when combined with (i)a poxviral vaccine targeting PSA48 and (ii) sipuleucel-T(ProvengeVR ), an autologous DC-based vaccine.49 These trialssuggest that docetaxel treatment has effects that go beyonddirectly inhibiting tumor growth.50 Our finding of enhancedCTL lysis following docetaxel treatment suggests that drug-induced cellular changes could be of clinical benefit in thetreatment of some types of tumors for which docetaxel isnot currently the standard of care, when combined withimmunotherapies. Furthermore, the enhanced CTL lysisobserved in a docetaxel-resistant cell line suggests that com-bining immunotherapy and chemotherapy may lead toimproved outcomes in patients who have previously failedchemotherapy alone.

Here, we show that treatment with a chemotherapeuticagent that does not induce ICD can nonetheless generate themolecular and immunologic hallmarks of immunogenic mod-ulation, expanding the set of chemotherapies that can beeffectively used in combination with immunotherapy.

AcknowledgementsThe authors thank Dr. Jeffrey Schlom for his helpful suggestions,Andressa Smith and Momodou Jammeh for ICD analysis, and MarionTaylor for excellent technical assistance. They also thank Bonnie L.Casey and Debra Weingarten for editorial assistance in the preparationof this article.

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chemotherapyinduced immunogenic modulation of tumor …to-5; erp57-speciﬁc mab to-2; and...

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